Protozoologica Acta Doi:10.4467/16890027AP.15.019.3216 Protozoologica
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Acta Protozool. (2015) 54: 233–240 www.ejournals.eu/Acta-Protozoologica ActA doi:10.4467/16890027AP.15.019.3216 Protozoologica Apolocystis proventus sp. nov. (Apicomplexa: Monocystinae) a New Species of Aseptate Gregarine from Egyptian Earthworms: Pheretima californica and Pheretima elongata (Annelida: Oligochaeta) Nadia RAMADAN, Samia FAWZY, Medhat ALI, Ahmed NIGM Department of Zoology, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt Abstract. A new acephaline gregarine is described from the earthworms Pheretima californica and Pheretima elongata. The gregarine was either embedded in the pharyngeal glandular tissue or found free in the coelomic fluid around the pharyngeal region in front of the crop. Adult trophozoites measured 48–65 µm in diameter and are mostly active with a wavy pellicle. Heterogeneity in the endoplasm of active trophozoites was observed. Gametocysts measured 56–81 µm in diameter, with a characteristic thick cyst wall. Navicular sporocysts mea- sured 5.8 ± 0.2 × 3.5 ± 0.4 µm, with small truncate plugs. Key words: Gregarine, Pheretima, Coelom, Apolocystis, Pharyngeal glands, Apicomplexa and Monocystinae. INTRODUCTION cystis by Cognett de Martiis (1923). Several species of Apolocystis were described (Bahatia and Setna 1926, Phillips and Mackinnon 1946, Ramadan 1969, Levine Monocystids belonging to the eugregarines are par- 1977, Segun 1978, Pradhan and Dasgupta 1983, Ar- asitic protozoa frequently infecting the seminal vesi- mendáriz and Gullo 2002, Bandyopadhyay et al. 2004, cles and coelomic cavities of many oligochaetes. Most Bhowmik et al. 2012). In this paper, a new species of published papers, which described those parasites, are Apolocystis is described with comparative comments taxonomic in nature or check lists of monocystid spe- on closely related species. cies along with their related hosts (Ruston 1959, Marek 1967, Segun 1971a, b, Levine 1977 and 1988, Pizl 1989a, b and Frolov 1991). All species belonging to MATERIALS AND METHODS genus Monocystis, characterized by round trophozoites with no polarity, were included into the genus Apolo- The host worms Earthworms were collected from Maghagha region (Menia Address for correspondence: Medhat Ali, Department of Zoology, Governorate) and the Zoological Gardens (Giza Governorate), Faculty of Science, Ain Shams University, Abbassia PO box 11566, Egypt. Specimens collected were stored into soil-filled plastic con- Cairo, Egypt; E-mail: [email protected]; Fax: 26842123 tainers and transferred live to the laboratory of invertebrates, depart- 234 N. Ramadan et al. ment of zoology, faculty of science, Ain shams university. Some Genus: Apolocystis (Cognetti de martiis 1923) earthworms were dissected, and the seminal vesicles were carefully Description: sub-spherical, oval, parasite is embed- removed and placed on a clean grease-free slide with a drop of 0.8% ded into the pharyngeal glandular tissue or free in the NaCl solution. A thin film of seminal fluid was pipette onto the slides and saved with glass cover slips. Wet mounts were examined coelomic fluid around the pharyngeal region in front of for living protozoans under a compound microscope. The coelomic the crop region. Adult trophozoites have no polar dif- fluid of the examined worms was withdrawn by a micropipette and ferentiation (48–65 µm in diameter). Most trophozoites then placed on clean slides with a drop of 0.8% NaCl solution and are active with wavy pellicle. Heterogeneity (irregular covered with glass cover slips for examination. After initial exam- distribution of paraglycogen granules) in the endoplasm ination of living protozoans, slides were semidried and fixed for of active trophozoites is observed. Gametocysts meas- 20 minutes in Schaudin’s fluid (saturated aqueous solution of mer- curic chloride and glacial acetic acid). Slides were then treated in ure 56–81 µm in diameter, with a characteristic thick ethyl alcohol for removal of excess of mercuric chloride. cyst wall. Navicular sporocysts measure 5.8 µm ± 0.2 × 3.5 µm ± 0.4, with small truncate plugs. Histological preparations Remarks: The parasite was found embedded in the For histological examination, sectioned tissues were prepared pharyngeal glands, (Fig. 1) free in the coelom, and in by fixing body segments of infected worms in Bouin’s fluid for front of the crop region of Pheretima californica and 24 hours. Specimens were then transferred to a mixture of 70% ethyl P. elongata. Growing trophozoites were mainly round alcohol and lithium carbonate, then dehydrated through an ascend- ing series of alcohols and cleared in terpineol for 3 days. Specimens or oval and had no apparent polarity. The pellicle exhib- were embedded in parablast wax and sectioned (5 µm thickness) on ited active short contractility waves which took many clean slides. Slides were then dewaxed in xylene, then rehydrated forms (Fig. 2). The endoplasm of the growing tropho- through a descending series of alcohols to distilled water. A subset zoites exhibited more heterogeneity than adults. Many of slides were transferred to Mallory I solution (1% Acid fuchsin) growing trophozoites had endoplasmic areas without for 15 seconds, rinsed in distilled water to differentiate red color of paraglycogen granules. In fresh preparations, the en- fuchsin for 10 seconds, treated with Phosphomolybdic acid (1%) for 1–5 minutes, then stained with Mallory II solution (equal parts of doplasm of some adult trophozoites exhibited an ir- 1% Aniline blue and 4% Orange G) for 2 minutes. Differentiation regular darker area (more aggregation of paraglycogen was done with 90% ethyl. Slides were then washed in distilled wa- granules) around the nucleus with narrow radiating ter, dehydrated in an ascending series of alcohols, cleared in xylene, extensions (Fig. 3). Paraglycogen granules were oval and mounted in Canada balsam. The other subset of slides were to elongate in shape with rounded ends and ranged stained in haematoxylin for 30 minutes, transferred to a tap water (1 minute), and counter stained in eosin. Slides were dehydrated in from 0.3 µm to 1.6 µm in length. The nucleus was de- ethyl alcohol, cleared and mounted. Photomicrographs were taken formed (irregular in shape) in most trophozoites due to by a Kodak digital camera (model 1450Z) attached to a compound the pressure exerted by the active internal streaming of microscope. the endoplasm. The nucleus was round, eccentric, and measured about 8–12 µm in diameter (Fig. 4). Karyo- somes were relatively large, eccentric and about 2.4– RESULTS 5.7 µm in diameter (Fig. 4). The adult trophozoites had no polar differentiation and measured about 48–65 µm in diameter (Table 1). Apolocystis proventus sp. nov. Gametocysts had sizes ranged from 56 to 81 μm in Phylum: Apicomplexa (Levine 1977) diameter (Fig. 5). The cyst wall is characterized by its Order: Eugregarinida (Leger 1900) considerable thickness. However, the morphological Family: Monocystidae (Butschli 1882) change of the parasite inside the cyst was hard to ob- Subfamily: Monocystinae Grassè, 1953 (= Mono- serve during progressive stages. In sectioned material, cystidae Bhatia, 1930) the cyst wall was deeply stained with Haematoxylin Figs 1–6. Apolocystis proventus a new greagarine from Egyptian earthworms. 1 – Section of the host worm showing a parasite in gametocyst stage (arrowhead) embedded in pharyngeal glands (PG), Mallory stain. Note-Insert showing a magnified gametocyst.2 – An active growing trophozoite showing the waves of contractility (arrowheads), fresh preparation. 3 – A growing trophozoite showing an irregular darker area (more aggregation of granules) around the nucleus (N) with narrow radiating extensions (white arrowhead), fresh preparation. 4 – Adult trophozoite showing the nucleus (N) and karyosome (K), fresh preparation. 5 – A mature gametocyst filled with sporocysts (S), fresh prepa- ration. 6 – A section of immature gametocyst showing a thick gametocyst wall (G W), Haematoxylin and eosin stains. A New Aseptate Gregarine from Egyptian Earthworms 235 236 N. Ramadan et al. Table 1. Average size (µm) of the observed Apolocystis proventus stages in coelom of Pheretima californica and P. elongata. Stage Average (µm) Range (µm) Standard deviation (± µm) Trophozoite – Cell 57.7 48–65 6.3 – Nucleus 10.3 8–12 1.7 – Karyosome 4 2.4–5.7 1.6 – Granule 0.9 0.3–1.6 0.6 Gametocyst – Diameter 73 56–81 5.9 Sporocyst – Length 5.8 5.6–6 0.2 – Width 3.5 3.1–3.9 0.4 and eosin (Fig. 6). Sporocysts are navicular, with small Fig. 7. A single sporocyst, note the two small flat plugs (P), fresh truncate plugs at both ends, and measured 5.8 ± 0.2 µm preparation. in length and 3.5 ± 0.4 µm in width (Fig. 7). DISCUSSION In the present parasite, the endoplasm of the grow- ing trophozoite exhibited more heterogeneity than that of the adult trophozoite. The growing trophozoites had The parasite described herein belongs to the genus areas of their endoplasm devoid of paraglycogen gran- Apolocystis. The spheroidal form and lack of polarity in ules; with progressive growth of the trophozoite and its trophozoites are evident characteristics. One species, more production of paraglycogen granules, the degree A. aggregata was described and named by Ramadan of heterogeneity in the endoplasm decreased. General- (1969), from two Egyptian earthworms Pheretima cali- ly, trophozoites related to Ramadan’s work were much fornica and Pheretima hawayana (Table 2). Apart from larger in its measurements (about double) than those of the difference in trophozoite size, and larger nuclei of the present gregarine. (Table 2) A. aggregata, and the gregarine describe herein, a spe- Within the range size of trophozoites of the present cial character was exhibited by A. aggregata. Young parasite, were those of A. beaufortii (Cognetti de Mar- trophozoites displayed the tendency to aggregate in tiis 1918) (50 μm); A. chotonagpurensis Bandyopad- groups consisting of 5 to 20 individuals. Moreover, the hyaye et al., 2004 (42 to 83 μm); A. minima (Boisson size and shape of the sporocysts in A. aggregata dif- 1957) Frolov 1991 (60 μm); and A. minuta Troisi, fered from these of the present species.